RESUMEN
Aldehydes in cigarette smoke (CS) impair mitochondrial function and reduce ciliary beat frequency (CBF), leading to diminished mucociliary clearance (MCC). However, the effects of aldehyde e-cigarette flavorings on CBF are unknown. The purpose of this study was to investigate whether cinnamaldehyde, a flavoring agent commonly used in e-cigarettes, disrupts mitochondrial function and impairs CBF on well-differentiated human bronchial epithelial (hBE) cells. To this end, hBE cells were exposed to diluted cinnamon-flavored e-liquids and vaped aerosol and assessed for changes in CBF. hBE cells were subsequently exposed to various concentrations of cinnamaldehyde to establish a dose-response relationship for effects on CBF. Changes in mitochondrial oxidative phosphorylation and glycolysis were evaluated by Seahorse Extracellular Flux Analyzer, and adenine nucleotide levels were quantified by HPLC. Both cinnamaldehyde-containing e-liquid and vaped aerosol rapidly yet transiently suppressed CBF, and exposure to cinnamaldehyde alone recapitulated this effect. Cinnamaldehyde impaired mitochondrial respiration and glycolysis in a dose-dependent manner, and intracellular ATP levels were significantly but temporarily reduced following exposure. Addition of nicotine had no effect on the cinnamaldehyde-induced suppression of CBF or mitochondrial function. These data indicate that cinnamaldehyde rapidly disrupts mitochondrial function, inhibits bioenergetic processes, and reduces ATP levels, which correlates with impaired CBF. Because normal ciliary motility and MCC are essential respiratory defenses, inhalation of cinnamaldehyde may increase the risk of respiratory infections in e-cigarette users.
Asunto(s)
Acroleína/análogos & derivados , Sistemas Electrónicos de Liberación de Nicotina , Aromatizantes/farmacología , Mitocondrias/efectos de los fármacos , Nicotina/farmacología , Acroleína/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Fumar/efectos adversosRESUMEN
OBJECTIVE: Mucociliary clearance sustains a baseline functionality and an "on demand" capability to upregulate clearance upon irritant exposure involving mucus hypersecretion and accelerated ciliary beat frequency (CBF) modulated by nitric oxide (NO). This study characterized these elements as well as cellular and exogenous NO concentrations subsequent to a single exposure to tobacco smoke (TS) or e-cigarette vapor (EV) on cultured human airway epithelium. MATERIALS AND METHODS: Air-liquid interface (ALI) airway epithelial cultures per nonsmoking human subjects were subjected to single TS or EV exposures. Measures of ciliary function and secretion were performed and cellular and exogenous NO concentrations under control and experimental conditions were assessed. RESULTS: Both TS and EV exposures resulted similar patterns of decline in CBF within 1 min of the completion of exposure followed by a gradual return often exceeding baseline within 1 h. Post-exposure examination of exposed cultures suggested morphologic differences in secretory function relative to controls. The relative NO concentrations of TS and EV chamber air were sharply different with EV NO being only slightly elevated relative to cellular NO production. DISCUSSION AND CONCLUSIONS: Epithelial remodeling and mucociliary dysfunction have been clearly associated with TS exposure. However, information contrasting epithelial structure/function following a single acute TS or EV exposure is limited. This study demonstrates a similar pattern of epithelial response to acute TS or EV exposure. Inasmuch as NO may contribute to an inflammatory milieu and generation of toxic metabolites, it is plausible that recurrent exposures over time may be contributory to chronic pathologies.
Asunto(s)
Sistemas Electrónicos de Liberación de Nicotina , Mucosa Nasal/efectos de los fármacos , Nicotiana , Humo/efectos adversos , Diferenciación Celular , Células Cultivadas , Cilios/efectos de los fármacos , Cilios/fisiología , Humanos , Microscopía Electrónica de Rastreo , Depuración Mucociliar , Mucosa Nasal/citología , Mucosa Nasal/metabolismo , Mucosa Nasal/ultraestructura , Óxido Nítrico/metabolismoRESUMEN
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ~60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.
Asunto(s)
Síndrome de Kartagener/genética , Proteínas Asociadas a Microtúbulos/genética , Mutación , Adulto , Preescolar , Exoma , Femenino , Genes Recesivos , Humanos , Masculino , Persona de Mediana Edad , Linaje , Isoformas de Proteínas , Análisis de Secuencia de ADNRESUMEN
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only â¼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders.
Asunto(s)
Antígenos de Superficie/genética , Cilios/genética , Trastornos de la Motilidad Ciliar/genética , Dineínas/genética , Proteínas de Unión al GTP/genética , Síndrome de Kartagener/genética , Mutación/genética , Adolescente , Adulto , Animales , Axonema/genética , Niño , Preescolar , Citoplasma/genética , Células Epiteliales/metabolismo , Exoma , Femenino , Humanos , Lactante , Masculino , Linaje , Fenotipo , Adulto Joven , Pez CebraRESUMEN
Primary ciliary dyskinesia (PCD) is caused when defects of motile cilia lead to chronic airway infections, male infertility, and situs abnormalities. Multiple causative PCD mutations account for only 65% of cases, suggesting that many genes essential for cilia function remain to be discovered. By using zebrafish morpholino knockdown of PCD candidate genes as an in vivo screening platform, we identified c21orf59, ccdc65, and c15orf26 as critical for cilia motility. c21orf59 and c15orf26 knockdown in zebrafish and planaria blocked outer dynein arm assembly, and ccdc65 knockdown altered cilia beat pattern. Biochemical analysis in Chlamydomonas revealed that the C21orf59 ortholog FBB18 is a flagellar matrix protein that accumulates specifically when cilia motility is impaired. The Chlamydomonas ida6 mutant identifies CCDC65/FAP250 as an essential component of the nexin-dynein regulatory complex. Analysis of 295 individuals with PCD identified recessive truncating mutations of C21orf59 in four families and CCDC65 in two families. Similar to findings in zebrafish and planaria, mutations in C21orf59 caused loss of both outer and inner dynein arm components. Our results characterize two genes associated with PCD-causing mutations and elucidate two distinct mechanisms critical for motile cilia function: dynein arm assembly for C21orf59 and assembly of the nexin-dynein regulatory complex for CCDC65.
Asunto(s)
Trastornos de la Motilidad Ciliar/genética , Glicoproteínas/genética , Síndrome de Kartagener/genética , Pez Cebra/genética , Animales , Chlamydomonas/genética , Cilios/genética , Análisis Mutacional de ADN/métodos , Dineínas/genética , Femenino , Humanos , Masculino , Mutación , Sistemas de Lectura Abierta , Planarias/genética , Proteoma/genéticaRESUMEN
RATIONALE: The relationship between clinical phenotype of childhood primary ciliary dyskinesia (PCD) and ultrastructural defects and genotype is poorly defined. OBJECTIVES: To delineate clinical features of childhood PCD and their associations with ultrastructural defects and genotype. METHODS: A total of 118 participants younger than 19 years old with PCD were evaluated prospectively at six centers in North America using standardized procedures for diagnostic testing, spirometry, chest computed tomography, respiratory cultures, and clinical phenotyping. MEASUREMENTS AND MAIN RESULTS: Clinical features included neonatal respiratory distress (82%), chronic cough (99%), and chronic nasal congestion (97%). There were no differences in clinical features or respiratory pathogens in subjects with outer dynein arm (ODA) defects (ODA alone; n = 54) and ODA plus inner dynein arm (IDA) defects (ODA + IDA; n = 18) versus subjects with IDA and central apparatus defects with microtubular disorganization (IDA/CA/MTD; n = 40). Median FEV1 was worse in the IDA/CA/MTD group (72% predicted) versus the combined ODA groups (92% predicted; P = 0.003). Median body mass index was lower in the IDA/CA/MTD group (46th percentile) versus the ODA groups (70th percentile; P = 0.003). For all 118 subjects, median number of lobes with bronchiectasis was three and alveolar consolidation was two. However, the 5- to 11-year-old IDA/CA/MTD group had more lobes of bronchiectasis (median, 5; P = 0.0008) and consolidation (median, 3; P = 0.0001) compared with the ODA groups (median, 3 and 2, respectively). Similar findings were observed when limited to participants with biallelic mutations. CONCLUSIONS: Lung disease was heterogeneous across all ultrastructural and genotype groups, but worse in those with IDA/CA/MTD ultrastructural defects, most of whom had biallelic mutations in CCDC39 or CCDC40.
Asunto(s)
Alelos , Proteínas del Citoesqueleto/genética , Síndrome de Kartagener/genética , Mutación , Proteínas/genética , Adolescente , Biomarcadores/sangre , Biopsia , Índice de Masa Corporal , Canadá , Niño , Preescolar , Femenino , Genotipo , Humanos , Síndrome de Kartagener/diagnóstico , Masculino , Fenotipo , Estudios Prospectivos , Índice de Severidad de la Enfermedad , Espirometría , Estados UnidosRESUMEN
RATIONALE: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous recessive disorder of motile cilia, but the genetic cause is not defined for all patients with PCD. OBJECTIVES: To identify disease-causing mutations in novel genes, we performed exome sequencing, follow-up characterization, mutation scanning, and genotype-phenotype studies in patients with PCD. METHODS: Whole-exome sequencing was performed using NimbleGen capture and Illumina HiSeq sequencing. Sanger-based sequencing was used for mutation scanning, validation, and segregation analysis. MEASUREMENTS AND MAIN RESULTS: We performed exome sequencing on an affected sib-pair with normal ultrastructure in more than 85% of cilia. A homozygous splice-site mutation was detected in RSPH1 in both siblings; parents were carriers. Screening RSPH1 in 413 unrelated probands, including 325 with PCD and 88 with idiopathic bronchiectasis, revealed biallelic loss-of-function mutations in nine additional probands. Five affected siblings of probands in RSPH1 families harbored the familial mutations. The 16 individuals with RSPH1 mutations had some features of PCD; however, nasal nitric oxide levels were higher than in patients with PCD with other gene mutations (98.3 vs. 20.7 nl/min; P < 0.0003). Additionally, individuals with RSPH1 mutations had a lower prevalence (8 of 16) of neonatal respiratory distress, and later onset of daily wet cough than typical for PCD, and better lung function (FEV1), compared with 75 age- and sex-matched PCD cases (73.0 vs. 61.8, FEV1 % predicted; P = 0.043). Cilia from individuals with RSPH1 mutations had normal beat frequency (6.1 ± Hz at 25°C), but an abnormal, circular beat pattern. CONCLUSIONS: The milder clinical disease and higher nasal nitric oxide in individuals with biallelic mutations in RSPH1 provides evidence of a unique genotype-phenotype relationship in PCD, and suggests that mutations in RSPH1 may be associated with residual ciliary function.
Asunto(s)
Proteínas de Unión al ADN/genética , Síndrome de Kartagener/genética , Mutación , Adolescente , Adulto , Niño , Cilios/fisiología , Análisis Mutacional de ADN , Exoma , Femenino , Estudios de Asociación Genética , Marcadores Genéticos , Pruebas Genéticas , Homocigoto , Humanos , Síndrome de Kartagener/fisiopatología , Modelos Lineales , Masculino , Persona de Mediana Edad , Mucosa Nasal/fisiología , Análisis de Secuencia de ADN , Adulto JovenRESUMEN
The nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent tobacco-specific carcinogen. We used an air-liquid interface epithelial cell culture system to model changes associated with NNK exposure relative to pathologies documented in human tobacco-related illnesses. Although in vitro systems exhibit certain limitations, they often offer accentuation of subtle pathologies. While the distribution of cell types in control cultures typically favors the ciliated cell phenotype, NNK-exposed cultures transitioned to non-ciliated cell phenotypes as well as reflecting features consistent with squamous metaplasia. We conclude that NNK impacts normal growth and differentiation of human airway epithelium in a short interval of time in vitro.
Asunto(s)
Carcinógenos/farmacología , Células Epiteliales/ultraestructura , Epitelio/metabolismo , Nicotiana/química , Nitrosaminas/farmacología , Butanonas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , FenotipoRESUMEN
Abstract Diagnosis of primary ciliary dyskinesia (PCD) by identification of dynein arm loss in transmission electron microscopy (TEM) images can be confounded by high background noise due to random electron-dense material within the ciliary matrix, leading to diagnostic uncertainty even for experienced morphologists. The authors developed a novel image analysis tool to average the axonemal peripheral microtubular doublets, thereby increasing microtubular signal and reducing random background noise. In a randomized, double-blinded study that compared two experienced morphologists and three different diagnostic approaches, they found that use of this tool led to improvement in diagnostic TEM test performance.
Asunto(s)
Dineínas Axonemales/ultraestructura , Interpretación de Imagen Asistida por Computador/métodos , Síndrome de Kartagener/diagnóstico , Microscopía Electrónica de Transmisión/métodos , Método Doble Ciego , Humanos , Reproducibilidad de los ResultadosRESUMEN
Primary ciliary dyskinesia (PCD) is a rare, autosomal recessive, genetically heterogeneous disorder characterized by ciliary dysfunction resulting in chronic oto-sino-pulmonary disease, respiratory distress in term neonates, laterality (situs) defects, and bronchiectasis. Diagnosis has traditionally relied on ciliary ultrastructural abnormalities seen by electron microscopy. Mutations in radial spoke head proteins occur in PCD patients with central apparatus defects. Advances in genetic testing have been crucial in addressing the diagnostic challenge. Here, we describe a novel splice-site mutation (c.921+3_6delAAGT) in RSPH4A, which leads to a premature translation termination signal in nine subjects with PCD (seven families). Loss-of-function was confirmed with quantitative ciliary ultrastructural analysis, measurement of ciliary beat frequency and waveform, and transcript analysis. All nine individuals carrying c.921+3_6delAAGT splice-site mutation in RSPH4A were Hispanic with ancestry tracing to Puerto Rico. This mutation is a founder mutation and a common cause of PCD without situs abnormalities in patients of Puerto Rican descent.
Asunto(s)
Efecto Fundador , Hispánicos o Latinos/genética , Síndrome de Kartagener/genética , Mutación , Proteínas/genética , Adolescente , Adulto , Alelos , Niño , Cilios/genética , Cilios/ultraestructura , Proteínas del Citoesqueleto , Femenino , Genotipo , Humanos , Masculino , Sitios de Empalme de ARN , Adulto JovenRESUMEN
We previously demonstrated that, in nasal epithelial cells (NECs) from smokers, methylation of an antiviral gene was associated with impaired antiviral defense responses. To expand these findings and better understand biological mechanisms underlying cigarette smoke (CS)-induced modifications of host defense responses, we aimed to compare DNA methylation of genes that may play a role in antiviral response. We used a two-tiered analytical approach, where we first implemented a genome-wide strategy. NECs from smokers differed in the methylation levels of 390 genes, the majority (84%) of which showed decreased methylation in smokers. Secondly, we generated an a priori set of 161 antiviral response-related genes, of which five were differentially methylated in NEC from smokers (CCL2, FDPS, GSK3B, SOCS3, and ULBP3). Assessing these genes at the systems biology level revealed a protein interaction network associated with CS-induced epigenetic modifications involving SOCS3 and ULBP3 signaling, among others. Subsequent confirmation studies focused on SOCS3 and ULBP3, which were hypomethylated and hypermethylated, respectively. Expression of SOCS3 was increased, whereas ULBP3 expression was decreased in NECs from smokers. Addition of the demethylating agent 5-Aza-2-deoxycytidine enhanced ULBP3 expression in NECs from smokers. Furthermore, infection of differentiated NECs with influenza virus resulted in significantly lower levels of ULBP3 in cells from smokers. Taken together, our findings show that genomic DNA methylation profiles are altered in NECs from smokers and that these changes are associated with decreased antiviral host defense responses, indicating that epigenenic dysregulation of genes such as SOCS3 and ULBP3 likely impacts immune responses in the epithelium.
Asunto(s)
Metilación de ADN , Células Epiteliales/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Mucosa Nasal/metabolismo , Fumar/efectos adversos , Fumar/fisiopatología , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Adulto , Azacitidina/análogos & derivados , Azacitidina/farmacología , Decitabina , Células Epiteliales/inmunología , Femenino , Proteínas Ligadas a GPI/biosíntesis , Proteínas Ligadas a GPI/metabolismo , Humanos , Virus de la Influenza A/inmunología , Gripe Humana/inmunología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Masculino , Mucosa Nasal/inmunología , Proteína 3 Supresora de la Señalización de Citocinas , TranscriptomaRESUMEN
CONTEXT: Investigations of cell/molecular level effects of in vivo exposure of airway mucosa of experimental animals to common irritant gases have demonstrated structural and physiological changes reflective of breaches in epithelial barrier function, presence of inflammatory cell infiltrate and compromised ciliary function. These experimental animal studies provided useful perspectives of plausible, but more subtle pathologic outcomes having relevance to lifestyle exposure to gaseous environmental irritants including tobacco smoke. METHODS: Freeze-fracture technology was applied to ultrastructural examination of large airway epithelium, with appropriate controls, from guinea pigs exposed to ozone and of nasal mucosa of human subjects exposed to ozone or sulfur dioxide, and nasal mucosa of active smokers. RESULTS: We documented substantive membrane structural changes to tight junctional complexes and cilia as well as an infiltrate of neutrophils into the surface mucosal layer in exposed animals. These patterns also were evident but not as pervasive among human subjects acutely exposed experimentally to irritant gases and those chronically exposed by their lifestyle to tobacco smoke. DISCUSSION: Our intent was to characterize respiratory tract mucosal membrane disorganization associated with high level acute irritant exposures in an experimental animal model and to evaluate evidence of similar but perhaps more subtle pathologic change associated with lower level experimental or lifestyle exposures. Our studies demonstrate continuity, albeit subtle, of pathologic change from high dosage experimental animal exposure to low dosage human exposures. CONCLUSIONS: This study represents the first report of ultrastructural airway epithelial membrane anomalies associated with lifestyle exposure to tobacco smoke irritants.
Asunto(s)
Mucosa Nasal/efectos de los fármacos , Oxidantes Fotoquímicos/toxicidad , Ozono/toxicidad , Fumar/efectos adversos , Dióxido de Azufre/toxicidad , Contaminación por Humo de Tabaco/efectos adversos , Animales , Biopsia , Membrana Celular/efectos de los fármacos , Membrana Celular/ultraestructura , Cotinina/orina , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Técnica de Fractura por Congelación/métodos , Cobayas , Humanos , Estilo de Vida , Masculino , Microscopía Electrónica de Transmisión , Mucosa Nasal/ultraestructura , Neutrófilos/efectos de los fármacos , Neutrófilos/patología , Fumar/sangre , Fumar/patología , Uniones Estrechas/efectos de los fármacos , Uniones Estrechas/ultraestructura , Contaminación por Humo de Tabaco/análisis , Tráquea/efectos de los fármacos , Tráquea/ultraestructuraRESUMEN
RATIONALE: Primary ciliary dyskinesia (PCD) is an autosomal recessive, genetically heterogeneous disorder characterised by oto-sino-pulmonary disease and situs abnormalities (Kartagener syndrome) due to abnormal structure and/or function of cilia. Most patients currently recognised to have PCD have ultrastructural defects of cilia; however, some patients have clinical manifestations of PCD and low levels of nasal nitric oxide, but normal ultrastructure, including a few patients with biallelic mutations in dynein axonemal heavy chain 11 (DNAH11). OBJECTIVES: To test further for mutant DNAH11 as a cause of PCD, DNAH11 was sequenced in patients with a PCD clinical phenotype, but no known genetic aetiology. METHODS: 82 exons and intron/exon junctions in DNAH11 were sequenced in 163 unrelated patients with a clinical phenotype of PCD, including those with normal ciliary ultrastructure (n=58), defects in outer and/or inner dynein arms (n=76), radial spoke/central pair defects (n=6), and 23 without definitive ultrastructural results, but who had situs inversus (n=17), or bronchiectasis and/or low nasal nitric oxide (n=6). Additionally, DNAH11 was sequenced in 13 subjects with isolated situs abnormalities to see if mutant DNAH11 could cause situs defects without respiratory disease. RESULTS: Of the 58 unrelated patients with PCD with normal ultrastructure, 13 (22%) had two (biallelic) mutations in DNAH11; and two patients without ultrastructural analysis had biallelic mutations. All mutations were novel and private. None of the patients with dynein arm or radial spoke/central pair defects, or isolated situs abnormalities, had mutations in DNAH11. Of the 35 identified mutant alleles, 24 (69%) were nonsense, insertion/deletion or loss-of-function splice-site mutations. CONCLUSIONS: Mutations in DNAH11 are a common cause of PCD in patients without ciliary ultrastructural defects; thus, genetic analysis can be used to ascertain the diagnosis of PCD in this challenging group of patients.
Asunto(s)
Dineínas Axonemales/genética , Cilios/ultraestructura , Trastornos de la Motilidad Ciliar/genética , Mutación , Adolescente , Adulto , Niño , Preescolar , Trastornos de la Motilidad Ciliar/diagnóstico , Trastornos de la Motilidad Ciliar/patología , Femenino , Genotipo , Humanos , Lactante , Masculino , Linaje , Fenotipo , Polimorfismo Genético , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
Epidemiologic evidence suggests that cigarette smoking is a risk factor for infection with influenza, but the mechanisms underlying this susceptibility remain unknown. To ascertain if airway epithelial cells from smokers demonstrate a decreased ability to orchestrate an influenza-induced immune response, we established a model using differentiated nasal epithelial cells (NECs) from nonsmokers and smokers, co-cultured with peripheral blood monocyte-derived dendritic cells (mono-DCs) from nonsmokers. NEC/mono-DC co-cultures were infected with influenza A virus and analyzed for influenza-induced immune responses 24 hours after infection. We observed that NECs from smokers, as well as mono-DCs co-cultured with NECs from smokers, exhibited suppressed influenza-induced, interferon-related proteins interferon regulatory factor-7, Toll-like receptor-3, and retinoic acid inducible gene-1, likely because of the suppressed production of IFNα from the NECs of smokers. Furthermore, NEC/mono-DC co-cultures using NECs from smokers exhibited suppressed concentrations of T-cell/natural killer cell chemokine interferon gamma-induced protein 10 (IP-10) after infection with influenza, indicating that NECs from smokers may skew early influenza-induced Th1 responses. In contrast, NEC/mono-DC co-cultures using NEC from smokers contained increased influenza-induced concentrations of the Th2 chemokine thymic stromal lymphopoeitin (TSLP). In addition, NECs from smokers cultured alone had increased influenza-induced concentrations of the Th2 chemokine thymus and activation-regulated chemokine (TARC). Using this model, we demonstrated that in the context of infection with influenza, NECs obtained from smokers create an overall cytokine microenvironment that suppresses the interferon-mediated Th1 response and enhances the TSLP-TARC-mediated Th2 response, with the potential to modify the responses of DCs. Smoking-induced alterations in the Th1/Th2 balance may play a role in developing underlying susceptibilities to respiratory viral infections, and may also promote the likelihood of acquiring Th2 proallergic diseases.
Asunto(s)
Células Dendríticas/inmunología , Células Epiteliales/inmunología , Gripe Humana/inmunología , Mucosa Nasal/inmunología , Fumar , Células TH1/inmunología , Células Th2/inmunología , Western Blotting , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo , Citocinas/genética , Citocinas/metabolismo , Células Dendríticas/metabolismo , Células Dendríticas/virología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Humanos , Virus de la Influenza A/patogenicidad , Gripe Humana/metabolismo , Gripe Humana/virología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/virología , Mucosa Nasal/metabolismo , Mucosa Nasal/virología , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1/metabolismo , Células TH1/virología , Células Th2/metabolismo , Células Th2/virologíaRESUMEN
OBJECTIVES: Nitric oxide (NO) is a reactive gas generated by inflammatory cells and mucosal epithelial cells of the nose and paranasal sinuses and is an important mediator in nonspecific host defense against infectious agents. However, NO also mediates physiologic events such as vasodilation, mucus hypersecretion, and mucosal disruption that are associated with inflammatory conditions, and it is a regulator of ciliary beat frequency. In the present study, we hypothesized that lifestyle exposure to tobacco smoke, whether through active smoking or by inadvertent exposure to secondhand tobacco smoke, would result in higher detectable levels of nasal NO (nNO) than are found in well-documented nonsmokers. METHODS: Nasal NO measurements were obtained concomitant with assays of urine cotinine from well-documented nonsmokers, active smokers, and individuals exposed by lifestyle to secondhand smoke. These parameters were statistically analyzed to determine whether increasing levels of tobacco smoke exposure yield higher concentrations of nNO. RESULTS: Our results and subsequent statistical analyses imply that active smokers who exhibit high urine cotinine levels exhibit significant increases in nNO levels in comparison to both nonsmokers and nonsmokers exposed to secondhand smoke. CONCLUSIONS: There is an increased level of nNO associated with tobacco smoke exposure that may contribute to the inflammatory processes characteristic of disease pathogenesis in smokers.
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Óxido Nítrico/metabolismo , Fumar/orina , Contaminación por Humo de Tabaco , Adulto , Pruebas Respiratorias , Cotinina/orina , Femenino , Humanos , Estilo de Vida , Masculino , Nariz/química , Adulto JovenRESUMEN
Smokers are more susceptible to respiratory viral infections, including influenza virus, but the mechanisms mediating this effect are unknown. To determine how epithelial cells contribute to the enhanced susceptibility seen in smokers, we established an in vitro model of differentiated nasal epithelial cells (NECs) from smokers, which showed enhanced mucin expression. The NECs from smokers responded to influenza infection with greater cytotoxicity, release of interleukin-6, and viral shedding than NECs from nonsmokers. Focusing on type I interferon (IFN) expression, we observed that influenza-infected NECs from smokers produced significantly less IFN-alpha than NECs from nonsmokers. Similarly, the expression of IRF7, a key transcription factor controlling the expression of IFN-alpha, was significantly decreased in influenza-infected and IFN-beta-stimulated NECs from smokers. Furthermore, our data indicate that the DNA methylation of the IRF7 gene and expression of the DNA (cytosine-5-)-methyltransferase 1 was enhanced in NECs from smokers. To confirm these findings in vivo, we initiated a study in which smoking and nonsmoking healthy volunteers were inoculated nasally with the live-attenuated influenza virus (LAIV) vaccine, and nasal biopsies were obtained before and after the administration of LAIV. The LAIV-induced expression of IRF7 was lower in the nasal epithelium from smokers, supporting our in vitro observations. These data demonstrate that infection with influenza results in the reduced expression of transcription factor IRF7 in NECs from smokers, and that these effects may be mediated by an epigenetic modification of the IRF7 gene, thus providing a potential mechanism rendering smokers more susceptible to respiratory virus infections.
Asunto(s)
Subtipo H3N2 del Virus de la Influenza A/patogenicidad , Gripe Humana/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Mucosa Nasal/metabolismo , Fumar/metabolismo , Adolescente , Adulto , Western Blotting , Estudios de Casos y Controles , Células Cultivadas , Citocinas/metabolismo , Metilación de ADN , Epigénesis Genética , Humanos , Técnicas para Inmunoenzimas , Gripe Humana/virología , Factor 7 Regulador del Interferón/genética , L-Lactato Deshidrogenasa/metabolismo , Mucosa Nasal/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
Mucociliary clearance, the physiological process by which mammalian conducting airways expel pathogens and unwanted surface materials from the respiratory tract, depends on the coordinated function of multiple specialized cell types, including basal stem cells, mucus-secreting goblet cells, motile ciliated cells, cystic fibrosis transmembrane conductance regulator (CFTR)-rich ionocytes, and immune cells1,2. Bronchiectasis, a syndrome of pathological airway dilation associated with impaired mucociliary clearance, may occur sporadically or as a consequence of Mendelian inheritance, for example in cystic fibrosis, primary ciliary dyskinesia (PCD), and select immunodeficiencies3. Previous studies have identified mutations that affect ciliary structure and nucleation in PCD4, but the regulation of mucociliary transport remains incompletely understood, and therapeutic targets for its modulation are lacking. Here we identify a bronchiectasis syndrome caused by mutations that inactivate NIMA-related kinase 10 (NEK10), a protein kinase with previously unknown in vivo functions in mammals. Genetically modified primary human airway cultures establish NEK10 as a ciliated-cell-specific kinase whose activity regulates the motile ciliary proteome to promote ciliary length and mucociliary transport but which is dispensable for normal ciliary number, radial structure, and beat frequency. Together, these data identify a novel and likely targetable signaling axis that controls motile ciliary function in humans and has potential implications for other respiratory disorders that are characterized by impaired mucociliary clearance.
Asunto(s)
Ciliopatías/inmunología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Depuración Mucociliar , Quinasas Relacionadas con NIMA/metabolismo , Adolescente , Adulto , Separación Celular , Niño , Ciliopatías/metabolismo , Células Epiteliales/metabolismo , Exoma , Femenino , Citometría de Flujo , Células HEK293 , Homocigoto , Humanos , Microscopía de Contraste de Fase , Microscopía por Video , Mutación , Fenotipo , Proteoma , Sistema Respiratorio , Tomografía Computarizada por Rayos X , Microtomografía por Rayos X , Adulto JovenRESUMEN
Primary ciliary dyskinesia is a genetically heterogeneous disorder of motile cilia. Most of the disease-causing mutations identified to date involve the heavy (dynein axonemal heavy chain 5) or intermediate(dynein axonemal intermediate chain 1) chain dynein genes in ciliary outer dynein arms, although a few mutations have been noted in other genes. Clinical molecular genetic testing for primary ciliary dyskinesia is available for the most common mutations. The respiratory manifestations of primary ciliary dyskinesia (chronic bronchitis leading to bronchiectasis, chronic rhino-sinusitis, and chronic otitis media)reflect impaired mucociliary clearance owing to defective axonemal structure. Ciliary ultrastructural analysis in most patients (>80%) reveals defective dynein arms, although defects in other axonemal components have also been observed. Approximately 50% of patients with primary ciliary dyskinesia have laterality defects (including situs inversus totalis and, less commonly, heterotaxy, and congenital heart disease),reflecting dysfunction of embryological nodal cilia. Male infertility is common and reflects defects in sperm tail axonemes. Most patients with primary ciliary dyskinesia have a history of neonatal respiratory distress, suggesting that motile cilia play a role in fluid clearance during the transition from a fetal to neonatal lung. Ciliopathies involving sensory cilia, including autosomal dominant or recessive polycystic kidney disease, Bardet-Biedl syndrome, and Alstrom syndrome, may have chronic respiratory symptoms and even bronchiectasis suggesting clinical overlap with primary ciliary dyskinesia.
Asunto(s)
Bronquitis Crónica/patología , Cilios/ultraestructura , Dineínas/genética , Síndrome de Kartagener/genética , Síndrome de Kartagener/patología , Animales , Bronquitis Crónica/etiología , Cilios/fisiología , Diagnóstico Diferencial , Humanos , Síndrome de Kartagener/complicaciones , Síndrome de Kartagener/diagnóstico , Ratones , Mutación/genéticaRESUMEN
The ciliated epithelium of the respiratory airways is one of the first vital systemic surfaces in contact with the ambient air. Ex vivo nasal epithelial ciliary beat frequency (CBF) at room temperature is on the order of 7-8 Hz but may be stimulated by irritant exposure. The upregulation of CBF in response to acute irritant exposure is generally considered to be a transient event with eventual return to baseline. However, studies of CBF dynamics in response to typical lifestyle exposures are limited. This study assessed nasal epithelial CBF among human subjects as a function of quantifiable lifestyle tobacco smoke exposure. Nasal epithelial biopsies were obtained from human subjects with well documented histories of tobacco smoke exposure. CBF was determined using a digital photometric technique and concurrent assays of nasal nitric oxide and urine cotinine and creatinine were performed. Mean CBF among active smokers and non-smokers exposed to environmental tobacco smoke (ETS) was elevated over non-smokers. Although there were dramatic differences in relative levels of tobacco smoke exposure, CBF values among tobacco smoke-exposed groups were comparable. Parallel in vitro studies of cultured nasal epithelium exposed to cigarette smoke condensate further supported these observations. These studies suggest that persistent elevation in nasal epithelial CBF is an early, subtle, physiologic effect associated with lifestyle tobacco smoke exposure. The molecular mechanisms that upregulate CBF may also create a cell molecular milieu capable of provoking the eventual emergence of more overt adverse health effects and the pathogenesis of chronic airway disease.